Unveilling genetic profiles and correlations of biofilm-associated genes, quorum sensing, and antibiotic resistance in Staphylococcus aureus isolated from a Malaysian Teaching Hospital

Background Staphylococcus aureus is a notorious multidrug resistant pathogen prevalent in healthcare facilities worldwide. Unveiling the mechanisms underlying biofilm formation, quorum sensing and antibiotic resistance can help in developing more effective therapy for S. aureus infection. There is a scarcity of literature addressing the genetic profiles and correlations of biofilm-associated genes, quorum sensing, and antibiotic resistance among S. aureus isolates from Malaysia. Methods Biofilm and slime production of 68 methicillin-susceptible S. aureus (MSSA) and 54 methicillin-resistant (MRSA) isolates were determined using a a plate-based crystal violet assay and Congo Red agar method, respectively. The minimum inhibitory concentration values against 14 antibiotics were determined using VITEK® AST-GP67 cards and interpreted according to CLSI-M100 guidelines. Genetic profiling of 11 S. aureus biofilm-associated genes and agr/sar quorum sensing genes was performed using single or multiplex polymerase chain reaction (PCR) assays. Results In this study, 75.9% (n = 41) of MRSA and 83.8% (n = 57) of MSSA isolates showed strong biofilm-forming capabilities. Intermediate slime production was detected in approximately 70% of the isolates. Compared to MSSA, significantly higher resistance of clindamycin, erythromycin, and fluoroquinolones was noted among the MRSA isolates. The presence of intracellular adhesion A (icaA) gene was detected in all S. aureus isolates. All MSSA isolates harbored the laminin-binding protein (eno) gene, while all MRSA isolates harbored intracellular adhesion D (icaD), clumping factors A and B (clfA and clfB) genes. The presence of agrI and elastin-binding protein (ebpS) genes was significantly associated with biofilm production in MSSA and MRSA isolates, respectively. In addition, agrI gene was also significantly correlated with oxacillin, cefoxitin, and fluoroquinolone resistance. Conclusions The high prevalence of biofilm and slime production among MSSA and MRSA isolates correlates well with the detection of a high prevalence of biofilm-associated genes and agr quorum sensing system. A significant association of agrI gene was found with cefoxitin, oxacillin, and fluoroquinolone resistance. A more focused approach targeting biofilm-associated and quorum sensing genes is important in developing new surveillance and treatment strategies against S. aureus biofilm infection. Supplementary Information The online version contains supplementary material available at 10.1186/s40001-024-01831-6.


Introduction
Staphylococcus aureus is one of the leading causes of severe bacterial infections which may lead to lifethreatening conditions, including sepsis, pneumonia, endocarditis, osteomyelitis, and implant-associated diseases [1][2][3].The emergence of antibiotic resistance in S. aureus has posed a significant impact on the treatment and infection control practices in hospitals worldwide [4,5].Methicillin-resistant S. aureus (MRSA), vancomycinresistant S. aureus (VRSA) and vancomycin-intermediate S. aureus (VISA) are among the pathogens listed as "High" priority in the World Health Organization (WHO)'s priority pathogens list for research and development of new antibiotics [6].
The ability of S. aureus to resist antimicrobials is further enhanced by the strategy of biofilm formation.Currently available antibiotics cannot eradicate biofilms, especially of ESKAPE pathogens, which includes MRSA [7].Several studies found no statistically significant difference in the biofilm formation among MRSA and MSSA strains [8,9].In contrary, a study reported that MRSA strains showed enhanced biofilm formation as compared to MSSA strains [10].Staphylococcal polysaccharide intercellular adhesin (encoded by icaABCD) [11], collagen-binding protein (cna), fibrinogen binding protein (fib), elastin binding protein (ebpS), laminin binding protein (eno), fibronectin binding proteins A and B (fnbA and fnbB), and clumping factors A and B (clfA and clfB) [12] have been reported to play important roles in S. aureus adherence, which is the first step in biofilm production.The icaABCD operon is also known for its function in slime production [13].
Quorum sensing is a mechanism, whereby bacterial cells communicate and coordinate their behaviours based on population density [14].The accessory gene regulator (agr) quorum-sensing system plays a key role in S. aureus pathogenesis, while the staphylococcal accessory regulator (sarA) gene is essential in controlling staphylococcal virulence factors [15].Both agr and sarA quorum sensing genes have been reported to regulate S. aureus biofilm formation [15][16][17][18].To date, four polymorphic agr types (agrI, agrII, agrIII, and agrIV) have been reported [19].
Previously, a high prevalence of icaADBC genes and varied occurrence of biofilm associated genes, i.e., cna (42.7-93%), fib (24.7-90%), ebps (11.1-100%), fnbA (0-100%) and fnbB (1.1-53.33%)have been reported in Malaysian S. aureus clinical isolates [20][21][22].The agr1 was the most prevalent type reported in Malaysian isolates of S.aureus, followed by agrII and agrIII; however, no agrIV was detected [21,23].Understanding differences in biofilm and slime production between MRSA and MSSA and the associated genetic elements contributes to a better understanding of the epidemiology and spread of S. aureus infections, further aiding in developing more targeted surveillance and treatment strategies.Hence, this study was performed to analyze biofilm and slime production of a collection of MSSA and MRSA clinical isolates and to investigate possible correlations between biofilm-associated genes and the agr/sar quorum sensing systems in relation to antibiotic resistance.

Congo red agar assay for determination of slime production
Bacterial slime production was determined qualitatively as described by Freeman et al. [27] and Thilakavathy et al. [28].Congo red agar was prepared using brain heart infusion (BHI) broth (37 g/L), sucrose (50 g/L), agar no.1 (10 g/L), and Congo red stain (0.8 g/L).Slime producers are expected to form black colonies with a dry, crystalline consistency, while non-slime producers form pink coloured colonies.Intermediate slime production is indicated by the growth of smooth blackishred colonies.The positive and negative control strains included in the test were Staphylococcus epidermidis ATCC ® 35984 ™ and Staphylococcus hominis ATCC ® 35982 ™ , respectively.

Bacterial genomic DNA extraction
Genomic DNA was extracted from overnight cultures of S. aureus in Luria-Bertani broth, using either MasterPure ™ Complete DNA and RNA Purification Kit (Lucigen, Middleton, WI, USA) or QIAamp DNA Mini Kit (Qiagen, Germany) following manufacturers' instructions.Amplification of the 16S rRNA gene from the bacterial DNA extract was performed to rule out the possibility of having PCR inhibitors, using universal oligonucleotide primers (27F and 1492R) as described by Gumaa et al. [29].

PCR detection of biofilm-associated genes
PCR profiling of bap, cna, icaA, and icaD genes was performed using singleplex PCR assays, while ebpS, eno, fnbA, clfA, clfB, fib, and fnbB genes were amplified using multiplex PCR assays as described by Tristan et al. and Vancraeynest et al. [30,31].The primers and PCR thermal cycling conditions are shown in Additional file 1: Table S2.sarA gene was amplified using sarAF and sarAR primers as described by Gowrishankar et al. [32].Meanwhile, agr typing (types I-IV) was performed using primers and amplification conditions as described by Shopsin et al. [19].The amplified products were then subjected to electrophoresis using 1% (w/v) agarose gel, pre-stained with nucleic acid staining dye (Bioteke Corporation, China).Sequence analyses were performed to confirm that correct genes were amplified.

Statistical analysis
Paired sample t tests were used to compare biofilm and slime production between MSSA and MRSA isolates.Pearson's Chi-square test was used to determine the correlation of antibiotic resistance with other parameters.Statistical analysis was performed using SPSS software version 20.0 (IBM, Armonk, USA).A p value of less than 0.05 was considered statistically significant.

Biofilm production of MRSA and MSSA isolates
Of the 122 S. aureus isolates tested, a majority (79.5%) were identified as strong biofilm producers.A total of 57 (83.8%) biofilm-producing isolates were MSSA and 41 (75.9%) isolates were MRSA (Table 2).In addition, 12.3% of S. aureus isolates were identified as moderate biofilm producers, 5.7% were identified as weak biofilm producers and 1.64% of strains did not produce biofilms.

Slime production of MRSA and MSSA isolates
Using Congo Red agar assay, most S. aureus isolates (72.1% MSSA and 72.2% MRSA isolates, respectively) were regarded as intermediate slime producers.There was no significant difference between MSSA and MRSA isolates in slime production (p = 0.19).Only 4 (6.0%)MSSA isolates demonstrated strong slime production after 24 h of incubation (Table 2).

Distribution of biofilm-associated genes and agr/sar quorum sensing genes in MSSA and MRS isolates
In this study, the successful amplification of the 16S rRNA gene from all S. aureus isolates indicated the absence of PCR inhibitors in the bacterial DNA extracts.The amplification of biofilm-associated genes from MSSA and MRSA isolates using various singleplex and  Strong slime producer 4 (5.9)0 multiplex PCR assays is shown in Additional file 1: Fig.

S1.
The presence of the intracellular adhesion A (icaA) gene was observed in all S. aureus isolates (100%).There was variability in the distribution of other biofilm-associated genes in MSSA and MRSA (Fig. 1).Overall, the intracellular adhesion A and D (icaA and icaD), lamininbinding protein (eno), clumping factors A and B (clfA and clfB), and fibronectin-binding protein A (fnbA) were the most prevalent biofilm-associated genes in S. aureus isolates, regardless of MSSA or MRSA.
Compared to MSSA, the detection rates of agrI, icaD, cna, clfA, and clfB genes were significantly higher in MRSA, while the fibronectin-binding protein B (fnbB) gene was absent in all MRSA isolates.All MSSA isolates harbored the laminin-binding protein (eno) gene, while all MRSA isolates harbored intracellular adhesion D (icaD), clumping factors A and B (clfA and clfB) genes.
Intriguingly, the bap gene (encoding biofilm matrix protein) was not amplified from any of the isolates.
The number of biofilm-associated genes detected in S. aureus varied from three to eleven, with most isolates having 10 genes (including 24 MSSA and 10 MRSA isolates).However, the number of genes detected from an isolate was not significantly associated with biofilm production (p = 0.299, Pearson's Chi-square, Table 3).Interestingly, the presence of agrI in MSSA (p = 0.018), and ebpS in MRSA isolates was significantly associated with biofilm production (p = 0.006) (Table 4).

Discussion
The treatment and management of S. aureus infection pose significant challenges and a big threat in healthcare settings worldwide due to the emergence  of antibiotic-resistant strains.In comparison with the Malaysia National Surveillance of Antimicrobial Resistance (NSAR) 2022 report [34], higher resistance rates to clindamycin (12.4% vs 5.9%), erythromycin (33.6% vs 9.9%) and gentamicin (13.1% vs 3.2%) were reported from a collection of clinical S. aureus isolates in this study.No linezolid-resistant strain was identified in this study, consistent with the very low percentage of linezolid resistance (0.4%) documented in the latest national report [34].So far, the highest linezolid resistance rate was reported in a previous NSAR study (2010) whereby 7.7% in MRSA and 3.3% in MSSA were linezolid resistant [1], while there have been no studies documenting S. aureus resistance to vancomycin in Malaysia [35,36].
In addition to antibiotic resistance, almost 80% of S. aureus isolates (MSSA and MRSA) in this study exhibited slime and biofilm production.However, no correlation was found between slime and biofilm production among staphylococcal isolates investigated in this study (Table 2).Similar observations have been reported for S. aureus human and animal isolates in earlier investigations [21,37].The lack of correlation between slime and biofilm production in S. aureus may be attributed to different measurement methods, i.e., Congo red agar method versus microtiter plate-based crystal violet assay, leading to disparities in the results.In addition, the complex nature of biofilm formation, possibly affected by bacterial genetic diversity, environmental factors, and regulatory mechanisms, may be attributed to the limited correlation between slime and biofilm production in S. aureus.
The most prevalent biofilm-associated genes detected in MRSA isolates in this study were intracellular adhesion A and D (icaA and icaD), laminin-binding protein (eno), clumping factors A and B (clfA and clfB), and fibronectin-binding protein A (fnbA), as shown in Fig. 1.The agrI, icaD, cna, clfA, and clfB genes were detected at significantly higher rates amongst MRSA isolates, while fnbB was detected at a significantly higher rate in MSSA isolates.The variability observed in the frequencies of biofilm-associated genes could be attributed to strain-to-strain difference [22,38], source of isolation [39], and geographical settings [40].Amongst the biofilm-associated genes, the elastin-binding protein (ebpS) gene has been significantly associated with biofilm production amongst MRSA isolates in this study (Table 3).Elastin-binding protein facilitates S. aureus-binding to elastin-rich tissues and promotes bacterial colonisation on mammalian tissues [41].It has been significantly associated with strong biofilm production in S. aureus food isolates in two previous studies [38,42].
The distribution of agr types is variable in S. aureus from different geographical regions [43].In this study, the most prevalent agr type identified from S. aureus isolates was agrI (56.7%), followed by agrIII (20.5%) and agrII (9.0%), while agrIV (0.8%) has a low occurrence rate.Remarkably, a significantly higher percentage of MRSA isolates in this study was found to harbor agrI, compared to MSSA.The presence of agrI has been significantly associated with biofilm production amongst MSSA isolates in this study (Table 3), corresponding well with another study using nonclinical isolates [42].Kawamura et al. [44] found that MRSA isolates haboring agrII have a significantly greater ability to produce biofilm, however; Usun Jones et al. [21] and Cha et al. [45] found no variation in MRSA biofilm production among different agr groups.The difference might be attributed to variations between strains, potentially resulting from microbial adaptation and geographical influences.
As the transcription of the agr locus (I-IV) is autoinducing peptide (AIP)-dependent, the differentiation of staphylococcal strains based on agr typing may provide further insights into the epidemiology and antibiotic resistance.Studies have shown that the mecA gene of MRSA indirectly activates AIPs which significantly affect biofilm production, quorum-sensing and virulence, and antibiotic resistance [17,18].As quorum sensing is higly influenced by cell density, high-density colonies can produce numerous small molecule signals, triggering downstream processes, such as virulence and antibiotic resistance mechanisms, which poses a threat to the host and antibiotic efficacy [46].Biofilm production has been reported to provide a niche for generation of antibiotic resistant subpopulations or persister cells through the exchange of genetic materials [47].Recent data demonstrated a significant correlation between agrI with cefoxitin and erythromycin resistance [48], as well as tetracycline, erythromycin, clindamycin, and ciprofloxacin resistance in S. aureus [43].Interestingly, a significant association was found between agrI with fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin) resistance (p < 0.05) for the first time in this study.In addition, the high resistance (75.9%) of MRSA against fluoroquinolones especially moxifloxacin, a fourth-generation fluoroquinolone, is alarming (Table 1).
Fluoroquinolone exposure has been identified as an increased risk factor for MRSA isolation and infection [49][50][51].The key mechanims to S. aureus fluoroquinolone resistance are through chromosomal point mutations in gyrA/B (DNA gyrase subunits), grlA/B (DNA topoisomerase IV subunits), and the promoter region of norA efflux pump [52].The accumulation of such mutations may be enhanced in biofilm producing agr1habouring strains, contributing to a high level of resistance to fluoroquinolones, as observed in the MRSA isolates in this study.However, more extensive studies are required to explore the linkage between agr1, biofilm production and fluoroquinolone resistance.
One of the limitation of this study is its confinement to a single-center setting and convenient sampling of S. aureus isolates, thus the ratio of MSSA to MRSA might not reflect the actual prevalence of multidrug resistant S. aureus in the local setting.For more comprehensive insights, future studies are recommended to include diverse sampling methods and multiple centers, to ensure a more representative analysis of the genetic diversity and prevalence of biofilm-associated genes in the Malaysian isolates.As the antibiotic susceptibility profiling of S. aureus isolates was limited to planktonic cells, future reserach should also include comprehensive assessment of antibiotic susceptibility within biofilm structures to enhance understanding of their impact on biofilm-associated S. aureus infections.In addition, the utilization of mec (SCCmec) typing would be beneficial for identifying distinct MRSA types and establishing correlations with other study variables.As conventional antibiotics do not work effectively against S. aureus biofilm infection, new therapeutic strategies and infection control practices are urgently needed.The genetic profiling of biofilm-associated genes and quorum sensing systems of S. aureus isolates has provided scientific foundation for developing a more targeted approach for surveillance, and treatment against biofilm infection in our clinical setting.

Conclusion
The emergence of multidrug-resistant S. aureus strains has been driven by the use of multiple antibiotic classes over the years.The high rates of resistance against clindamycin, erythromycin, and fluoroquinolones as reported in this study have called for more judicious use of antibiotics for treatment of MRSA infection in this region.More importantly, the identification of prevalent biofilmassociated genes and agr types associated with antibiotic resistance in this study has shed valuable genetic insights into S. aureus biofilm formation, which are important to tailor more focused surveillance and treatment strategies against S. aureus biofilm infection in our setting.

Table 1
Antibiotic susceptibility profiles of S. aureus isolates investigated in this study a 1 (1.5%) were inducible resistant; b 23 (42.6%) were inducible resistant; c missing information for 10 MSSA isolates, d p < 0.05 indicates significant difference between MSSA and MRSA, -: not applicable

Table 2
Distribution of biofilm and slime producers among MRSA and MSSA isolates *p values were determined using t test, with p > 0.05 indicating no significant difference between groups Biofilm/slime production No. (%) isolates p value* MSSA (n = 68) MRSA (n = 54)

Table 3
Cross-tabulation between the number of S. aureus biofilm-associated genes and biofilm production *p value were determined using Pearson's Chi-

Table 4
Correlations between agr, sarA, and biofilm-associated genes, with biofilm production in 68 MSSA and 54 MRSA isolates investigated in this study *p < 0.05 indicates significant difference between groups; -: not applicable

Table 5
Association between the presences of agrI with antibiotic susceptibility in S. aureus clinical isolatesLinezolid and vancomycin were excluded from the analysis as all S. aureus isolates were susceptible to these antibiotics.S: susceptible; R: resistant; IR: inducible resistant; IN: intermediate, *p < 0.05 indicates significant difference between groups